The anti-apoptotic Bcl-2 family members, Bcl-2 and Bcl-xL, are over-expressed in many cancer cells, ultimately leading to cell immortality and resistance to the conventional cancer therapies that trigger the apoptotic pathway. Therefore, we believe developing small molecule compounds that specifically target these anti-apoptotic proteins will reduce the cellular resistance to cancer therapies. The objectives are to develop and synthesize novel, highly potent and selective small molecule inhibitors to Bcl-xL and Bcl-2 and test them in vitro and in vivo for their therapeutic potential in cancer cells. Part of this proposal will complement the others by providing the atomic detailed analysis of inhibitor binding needed to increase potency and selectivity of the small molecule compounds toward Bcl-2 and BclxL. For our structural studies, we will employ the complimentary techniques of NMR and X-ray crystallography. To accomplish our goal, we propose to do the following four Specific Aims: Aim 1: Confirmation of inhibitor binding to the BH3 binding site of Bcl-xL and Bcl-2 using NMR screening methods; Aim 2: Determination of three-dimensional structures of Bcl-2 and Bcl-xL complexed with inhibitors by multidimensional NMR methods; Aim 3: Determination of high-resolution X-ray crystal structures of potent small molecule inhibitors in complex with Bcl-xL; Aim 4.: Analysis of Bcl-2 and Bcl-xL residues crucial for inhibitor binding through site-directed mutagenesis and biochemical binding studies. Structural studies of human BclxL and Bcl-2 in complex with representatives from each class of compounds will aid in identifying residues crucial for inhibitor binding and provide the foundation for designing new inhibitors within each class. Mutational analysis of non-conserved residues in the BH3 binding groove as well as residues involved in the structural stability of the binding site may offer insight into the protein's specificity for inhibitors. These studies in conjunction with the structural determination of Bcl-xL and Bcl-2, in complex with inhibitors, will aid in identifying key residues for inhibitor binding and provide the foundation for designing new inhibitors having greater specificity toward these individual targets.